Objectives: The main objectives of this proposal are to: (1) define how oncogenic EGFR/MAPK signal transduction influences the expression of MHC molecules and, (2) characterize the impact of therapies that target the EGFR and enzymes in the MAPK pathway on anti-tumor immune responses and the response to immunotherapy. Research Plan: In Aim 1, we will use well-characterized cell lines to model human cancers driven by EGFR over-expression, oncogenic RAS mutations, and oncogenic BRAF mutations. These cell lines will be used to determine how oncogenic EGFR/MAPK signal transduction and the medications developed to combat this signaling, impact the transcriptional and epigenetic regulation of MHC class I expression. In Aim 2, we will couple these same medications with relevant murine tumor models to determine how these medications impact in vivo immune responses to tumor challenge, the expression of relevant immune system genes, and the response to immunotherapies such as immune checkpoint blockade and therapeutic vaccination. Methods: Using the above models, we will combine EGFR, BRAFV600E, and MEK1/2 inhibitor treatment with chromatin immunoprecipitation experiments to define the impact of EGFR/MAPK signaling on the recruitment of transcription factors and the placement of epigenetic marks at the MHCI promoter. Using reporter assays, we will define regions within MHC class I promoters that are responsive to EGFR/MAPK signaling. To explore more complex regulatory mechanisms, we will investigate how EGFR/MAPK signaling impacts the function of an enhancer-blocking DNA element known to regulate MHC expression. We will also determine how EGFR/MAPK signaling impacts the recruitment/binding of CTCF, a protein that regulates MHC expression by mitigating complex chromatin interactions, to sites within the MHC. For our in vivo studies, we will use therapy- responsive and non-responsive tumor models to determine how EGFR/MAPK inhibitors impact tumor growth, the generation of tumor-reactive and antigen-specific CD8+ T cells, and the tumor microenvironment using flow cytometry, functional cytotoxic T lymphocyte (CTL) assays, RNA analysis and other approaches. We will explore the novel hypothesis that these medications have clinically relevant tumor-dependent and tumor- independent effects on host anti-tumor immunity that can be used to enhance the response to immunotherapy. Clinical Relevance: Inhibitors of the EGFR and MAPKs are already used to treat Veterans with advanced cancer with variable response rates. To help integrate these medications with new and developing therapies that are immune based, it will be crucial to have a sophisticated understanding of how these kinase inhibitors influence immune events in vivo. Indeed, despite the fact these inhibitors are already being combined with immunotherapies in clinical trials for those with advanced cancer, their impact on the immune system is largely undefined. By characterizing how these medications influence immune responses, we can develop a rational framework to optimize the use of these medications alone and in combination with immune-based therapy. In addition, by defining the mechanisms that govern EGFR/MAPK-mediated MHC repression, new approaches can be developed to disrupt the MHCI down regulation and resulting tumor cell immune escape that occurs commonly in cancer.